Integrins are a class of cellular receptors known to bind extracellular matrix proteins, and therefore mediate cell-cell and cell-extracellular matrix interactions, referred generally to as cell adhesion events. However, although many integrins and their respective ligands are described in the literature, the biological function of many of the integrins remains elusive. The integrin receptors constitute a family of proteins with shared structural characteristics of noncovalent heterodimeric glycoprotein complexes formed of α and β subunits.
The vitronectin receptor, named for its original characteristic of preferential binding to vitronectin, is now known to refer to three different integrins, designed αvβ1, αvβ3 and αvβ5. Horton, Int. J. Exp. Pathol., 71:741–759 (1990). αvβ1 binds fibronectin and vitronectin. αvβ3 binds a large variety of ligands, including fibrin, fibrinogen, laminin, thrombospondin, vitronectin, von Willebrand's factor, osteospontin and bone sialoprotein I. αvβ5 binds vitronectin. The specific cell adhesion roles these three integrins play in the many cellular interactions in tissues are still under investigation. However, it is clear that there are different integrins with different biological functions as well as different integrins and subunits having shared biological specificities.
One important recognition site in a ligand for many integrins is the arginine-glycine-aspartic acid (RGD) tripeptide sequence. RGD is found in all of the ligands identified above for the vitronectin receptor integrins. This RGD recognition site can be mimicked by polypeptides (“peptides”) that contain the RGD sequence, and such RGD peptides are known inhibitors of integrin function. It is important to note, however, that depending upon the sequence and structure of the RGD peptide, the specificity of the inhibition can be altered to target specific integrins.
For discussions of the RGD recognition site, see Pierschbacher et al., Nature, 309:30–33 (1984), and Pierschbacher et al., Proc. Natl. Acad. Sci. USA, 81:5985–5988 (1984). Various RGD polypeptides of varying integrin specificity have also been described by Grant et al., Cell, 58:933–943 (1989), Cheresh, et al., Cell, 58:945–953 (1989), Aumailley et al., FEBS Letts., 291:50–54 (1991), and Pfaff et al., J. Biol. Chem., 269:20233–20238 (1994), and in U.S. Pat. Nos. 4,517,686, 4,578,079, 4,589,881, 4,614,517, 4,661,111, 4,792,525, 4,683,291, 4,879,237, 4,988,621, 5,041,380 and 5,061,693.
Angiogenesis, also referred to as neovascularization, is a process of tissue vascularization that involves the growth of new developing blood vessels into a tissue. The process is mediated by the infiltration of endothelial cells and smooth muscle cells. The process is believed to proceed in any one of three ways: 1) The vessels can sprout from pre-existing vessels; 2) De novo development of vessels can arise from precursor cells (vasculogenesis); or 3) Existing small vessels can enlarge in diameter. Blood et al., Bioch. Biophys. Acta, 1032:89–118 (1990). Vascular endothelial cells are known to contain at least five RGD-dependent integrins, including the vitronectin receptor (αvβ3 or αvβ5), the collagen Types I and IV receptor (α1β1), the laminin receptor (α2β1), the fibronectin/laminin/collagen receptor (α3β1) and the fibronectin receptor (α5β1). Davis et al., J. Cell. Biochem., 51:206–218 (1993). The smooth muscle cell is known to contain at least six RGD-dependent integrins, including α5β1, αvβ3 and αvβ5.
Angiogenesis is an important process in neonatal growth, but is also important in wound healing and in the pathogenesis of a large variety of clinically important diseases including tissue inflammation, arthritis, psoriasis, cancer, diabetic retinopathy, macular degeneration and other neovascular eye diseases. These clinical entities associated with angiogenesis are referred to as angiogenic diseases. Folkman et al., Science, 235:442–447 (1987). Angiogenesis is generally absent in adult or mature tissues, although it does occur in wound healing and in the corpus luteum growth cycle. See, for example, Moses et al., Science, 248:1408–1410 (1990).
Inhibition of cell adhesion in vitro using monoclonal antibodies immunospecific for various integrin α or β subunits have implicated the vitronectin receptor αvβ3 in cell adhesion of a variety of cell types including microvascular endothelial cells. Davis et al., J. Cell. Biol., 51:206–218 (1993). In addition, Nicosia et al., Am. J. Pathol., 138:829–833 (1991), described the use of the RGD peptide, GRGDS, to inhibit the in vitro formation of “microvessels” from rat aorta cultured in collagen gel.
However, the inhibition of formation of “microvessels” in vitro in collagen gel cultures is not a model for inhibition of angiogenesis in a tissue because it is not shown that the microvessel structures are the same as capillary sprouts or that the formation of the microvessel in collagen gel culture is the same as new-vascular growth into an intact tissue, such as arthritic tissue, tumor tissue or disease tissue where inhibition of angiogenesis is desirable.
The role of αvβ3 in angiogenesis was recently confirmed. See, Brooks, et al. Science, 264:569–571 (1994). The integrin was shown to be expressed on blood vessels in human wound granulation tissue but not in normal skin. Monoclonal antibodies against the αvβ1 receptor inhibited angiogenesis induced by the growth factors (cytokines) basic fibroblast growth factor (bFGF) and tumor necrosis factor-α (TNF-α), as well as by melanoma fragments. However, the antagonists only inhibited new and not preexisting vessels. In addition, specific linear and cyclic RGD-containing peptides were also shown to inhibit neovascularization.
It has been proposed that inhibition of angiogenesis would be a useful therapy for restricting tumor growth. Inhibition of angiogenesis has been proposed by (1) inhibition of release of “angiogenic molecules” such as bFGF (basic fibroblast growth factor), (2) neutralization of angiogenic molecules, such as by use of anti-bFGF antibodies, and (3) inhibition of endothelial cell response to angiogenic stimuli. This latter strategy has received attention, and Folkman et al., Cancer Biology, 3:89–96 (1992), have described several endothelial cell response inhibitors, including collagenase inhibitor, basement membrane turnover inhibitors, angiostatic steroids, fungal-derived angiogenesis inhibitors, platelet factor 4, thrombospondin, arthritis drugs such as D-penicillamine and gold thiomalate, vitamin D3 analogs, alpha-interferon, and the like that might be used to inhibit angiogenesis. For additional proposed inhibitors of angiogenesis, see Blood et al., Bioch. Biophys. Acta., 1032:89–118 (1990), Moses et al., Science, 248:1408–1410 (1990), Ingber et al., Lab. Invest., 59:44–51 (1988), and U.S. Pat. Nos. 5,092,885, 5,112,946, 5,192,744, and 5,202,352.
However, the role of the integrin αvβ5 in angiogenesis has neither been suggested or identified until the present invention nor have any of the inhibitors of angiogenesis described in the foregoing references been targeted at inhibition of αvβ5. Moreover, no references, other than the present invention, have implicated the αvβ5 integrin in neovascularization, particularly that induced by the growth factors, vascular endothelial growth factor (VEGF), transforming growth factor-α (TGF-α) and epidermal growth factor (EGF).
Although the numbers of growth factors involved in the control of angiogenesis are limited, different levels of control of the process exist for conversion of a quiescent state to a neovascular state. See, D'Amore, Investigative Ophthal. Visual Sci., 35:3974–3979 (1994). While some growth factors involved in angiogenesis are regulated at the synthesis level, others are regulated by the state of activation. These cellular events occur as a quiescent vessel undergoes neovascularization following injury or ischemia.
VEGF, in particular, is thought to be a major mediator of angiogenesis in a primary tumor and in ischemic ocular diseases. For review, see Folkman, Nature Medicine, 1:27–31 (1995). VEGF is a 46 kilodalton (kDa) homodimer that is an endothelial cell-specific angiogenic (Ferrara et al., Endocrin. Rev., 13:18–32 (1992)) and vasopermeable factor (Senger et al., Cancer Res. 46:5629–5632 (1986)) that binds to high-affinity membrane-bound receptors with tyrosine kinase activity (Jakeman et al., J. Clin. Invest., 89:244–253 (1992)).
Activation of receptor tyrosine kinases has recently been shown to promote integrin-dependent cell migration on extracellular matrix proteins. In particular, Klemke et al., J. Cell Biol., 127:859–866 (1994) have implicated the EGF receptor (EGFR) tyrosine kinase in promoting cell motility but not adhesion of FG human pancreatic carcinoma cells on vitronectin using the αvβ5 integrin. The authors provide direct evidence that occupation of EGFR with the EGF ligand activates the tyrosine kinase activation of the EGFR that ultimately stimulates a protein kinase C (PKC)-dependent pathway leading to the induction of αvβ5-dependent cell migration of vitronectin substrate on which the cells are normally unable to migrate. Thus, the Klemke et al. findings provide evidence for correlating the presence of cytokines, specifically EGF, with integrin activity in cell migration. Activation of PKC has been shown to be involved in the regulation of angiogenesis in the chick chorioallantoic membrane model system. See, Tsopanoglou et al., J. Vasc. Res. 30:202–208 (1993). The authors identified specific activators and inhibitors of PKC that respectively stimulated and inhibited angiogenesis in the model system.
However, neither Klemke et al. nor Tsopanoglou et al. discussed above describe the role of cytokines and expression and/or activation of the αvβ5 integrin in promoting angiogenesis in various conditions and disease states and inhibition thereof with αvβ5-specific antagonists.
Recent experimental evidence has shown in a monkey model system of eye disease that retinal ischemia induced by retinal vein occlusion resulted in a rapid rise of VEGF in the aqueous chambers of the eye. This rise coincided with the iris neovascularization that was observed as described by Miller et al., Am. J. Path., 145:574–584 (1994). Additional data in an mouse model system of proliferative retinopathy in which hypoxia is induced, VEGF messenger RNA was shown to increase within 6–12 hours of relative hypoxia that remained elevated until neovascularization developed. As the new blood vessels declined, so did the VEGF expression as described by Pierce et al., Proc. Natl. Acad. Sci., USA, 92:905–909 (1995).
Thus, the recent data as demonstrated in animal models of ischemia have correlated the induction of VEGF with that of ischemia followed by neovascularization. VEGF, as well as other growth factors, have also been implicated in other conditions and disease states involving neovascularization as reviewed by Folkman, Nature Medicine, 1:27–31 (1995).
The Folkman et al. reference also summarizes the current clinical approaches used to control undesirable angiogenesis. Patients in clinical trials have received therapeutic treatments with angiogenic inhibitors including platelet factor 4, a fumagillin-derivative, carboxy-aminotriazole, and the like. However, no references or current therapeutic references correlate the expression of 60vβ5 with angiogenesis, particularly that induced by VEGF. Thus, prior to the present invention, no one has described nor utilized a therapeutic regimen with αvβ5 antagonists to control angiogenesis in a tissue undergoing angiogenesis correlated with the presence and activation of αvβ5.
Therefore, other than the studies reported here on αvβ3 and the relationship with growth factors to angiogenesis. Applicants are unaware of any other demonstration that angiogenesis could be inhibited in a tissue using inhibitors of αvβ5-mediated cell adhesion. In particular, it has never been previously demonstrated that αvβ5 function is required for angiogenesis in a tissue or that αvβ5 antagonists can inhibit angiogenesis in a tissue, particularly in ocular neovascular diseases.